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PDF file, 267KB, Supplementary figure 1. a) Heat maps of RKIP, EZH2, Snail and E-cad expression profiles obtained by interrogating publicly available DNA microarray expression datasets (33). The abbreviations are as follows: NAP: normal adjacent prostate tissue, BPH: benign prostate hyperplasia, PCA: prostate cancer adenocarcinoma, and MPC: metastatic prostate carcinoma. Rows correspond to individual genes and columns represent individual patients. Color density is arranged in order from greatest decrease in expression at left (red) to greatest increase at right (light yellow). EZH2 and Snail mRNA are strongly expressed in MPC, in contrast to RKIP and E-cad. b) DU145 or MDA-MB231 cells were infected with indicated siRNA expressing retroviruses. The siRNA expressing cells were assayed for their ability to proliferate in growth medium containing 0.3% agar and the formation of multi-cellular colonies. Colonies were stained with MTT and quantified after 21 days from plates in triplicate (lower panel). Data shown are representative of three independent experiments and are expressed as mean colony number per plate +/- s.d. c) The effect of the indicated siRNA on DU145 cells proliferation was assayed by direct cell counting. Quiescent cells were counted by coulter counter at the indicated times after stimulation with 20% FBS. Data shown are representative of three independent experiments.

Raf-1 kinase inhibitor protein was first identified as a negative regulator of the Raf signaling pathway. Subsequently, it was shown to have a causal role in containing cancer progression and metastasis. Early studies suggested that RKIP blocks cancer progression by inhibiting the Raf-1 pathway. However, it is not clear if the RKIP tumor and metastasis suppression function involve other targets. In addition to the Raf signaling pathway, RKIP has been found to modulate several other signaling pathways, affecting diverse biological functions including immune response. Recent advances in medicine have identified both positive and negative roles of immune response in cancer initiation, progression and metastasis. It is possible that one way that RKIP exerts its effect on cancer is by targeting an immune response mechanism. Here, we provide evidence supporting the causal role of tumor and metastasis suppressor RKIP in downregulating signaling pathways involved with immune response in breast cancer cells and discuss its potential ramification on cancer therapy.

Raf kinase inhibitor protein (RKIP) is a multifunctional modulator of intracellular signal transduction. Although most of its functions have been considered cytosolic, we show here that the localization of RKIP is primarily nuclear in both growing and quiescent Madin-Darby canine kidney epithelial cells and in Cal-51 and BT-20 human breast cancer cells. We have identified a putative bipartite nuclear localization signal (NLS) in RKIP that maps to the surface of the protein surrounding a known regulatory region. Like classical NLS sequences, the putative NLS of RKIP is rich in arginine and lysine residues. Deletion of and point mutations in the putative NLS lead to decreased nuclear localization. Point mutation of all the basic residues in the putative NLS of RKIP particularly strongly reduces nuclear localization. We found consistent results in reexpression experiments with wildtype or mutant RKIP in RKIP-silenced cells. A fusion construct of the putative NLS of RKIP alone to a heterologous reporter protein leads to nuclear localization of the fusion protein, demonstrating that this sequence alone is sufficient for import into the nucleus. We found that RKIP interacts with the nuclear transport factor importin α in BT-20 and MDA-MB-231 human breast cancer cells, suggesting importin-mediated active nuclear translocation. Taken together, these findings suggest that a bipartite NLS in RKIP interacts with importin α for active transport of RKIP into the nucleus and that this process may be involved in the regulation of mitotic progression. Evaluating the biological function of nuclear localization of RKIP, we found that the presence of the putative NLS is important for the role of RKIP in mitotic checkpoint regulation in MCF-7 human breast cancer cells.

Tumor microenvironment (TME) is the immediate environment where cancer cells reside in a tumor. It is composed of multiple cell types and extracellular matrix. Microenvironments can be restrictive or conducive to the progression of cancer cells. Initially, microenvironments are suppressive in nature. Stepwise accumulation of mutations in oncogenes and tumor suppressor genes enables cancer cells to acquire the ability to reshape the microenvironment to advance their growth and metastasis. Among the many genetic events, the loss-of-function mutations in tumor suppressor genes play a pivotal role. In this review, we will discuss the changes in TME and the ramifications on metastasis upon altered expression of tumor metastasis suppressor gene RKIP in breast cancer cells.

The role of RhoA GTPases in breast cancer tumorigenesis and metastasis is unclear. Early studies within which mutations in RhoA were designed based on cancer-associated mutations in Ras supported an oncogene role for RhoA. However, recent whole-genome sequencing studies of cancers raised the possibility that RhoA may have a tumor suppression function. Here, using a syngeneic triple negative breast cancer murine model we investigated the physiological effects of reduced RhoA expression on breast cancer tumorigenesis and metastasis. RhoA knockdown had no effect on primary tumor formation and tumor proliferation, concurring with our in vitro findings where reduced RhoA had no effect on breast cancer cell proliferation and clonogenic growth. In contrast, primary tumors with RhoA knockdown efficiently invaded sentinel lymph nodes and significantly metastasized to lungs compared to control tumors. Mechanistically, the current study demonstrated that this is achieved by promoting a pro-tumor microenvironment, with increased cancer-associated fibroblasts and macrophage infiltration, and by modulating the CCL5-CCR5 and CXCL12-CXCR4 chemokine axes in the primary tumor. To our knowledge, this is the first such mechanistic study in breast cancer showing the ability of RhoA to suppress chemokine receptor expression in breast tumor cells. Our work suggests a physiological lung and lymph node metastasis suppressor role for RhoA GTPase in breast cancer.

One third of newly diagnosed breast cancers in the US are early-stage lesions. The etiological understanding and treatment of these lesions have become major clinical challenges. Because breast cancer risk factors are often linked to aberrant nitric oxide (NO) production, we hypothesized that abnormal NO levels might contribute to the formation of early-stage breast lesions. We recently reported that the basal level of NO in the normal breast epithelia plays crucial roles in tissue homeostasis, whereas its reduction contributes to the malignant phenotype of cancer cells. Here, we show that the basal level of NO in breast cells plummets during cancer progression due to reduction of the NO synthase cofactor, BH4, under oxidative stress. Importantly, pharmacological deprivation of NO in prepubertal to pubertal animals stiffens the extracellular matrix and induces precancerous lesions in the mammary tissues. These lesions overexpress a fibrogenic cytokine, TGFβ, and an oncogene, ERBB2, accompanied by the occurrence of senescence and stem cell-like phenotype. Consistently, normalization of NO levels in precancerous and cancerous breast cells downmodulates TGFβ and ERBB2 and ameliorates their proliferative phenotype. This study sheds new light on the etiological basis of precancerous breast lesions and their potential prevention by manipulating the basal NO level.

Raf-1 kinase inhibitor protein was initially discovered as a physiological kinase inhibitor of the MAPK signaling pathway and was later shown to suppress cancer cell invasion and metastasis. Yet, the molecular mechanism through which RKIP executes its effects is not completely defined. RhoA has both a pro- and anti-metastatic cell-context dependent functions. Given that Rho GTPases primarily function on actin cytoskeleton dynamics and cell movement regulation, it is possible that one way RKIP hinders cancer cell invasion/metastasis is by targeting these proteins. Here we show that RKIP inhibits cancer cell invasion and metastasis by stimulating RhoA anti-tumorigenic functions. Mechanistically, RKIP activates RhoA in an Erk2 and GEF-H1 dependent manner to enhance E-cadherin membrane localization and inhibit CCL5 expression.

Recent high-throughput-sequencing of cancer genomes has identified oncogenic mutations in the BRaf genetic locus as one of the critical events in melanomagenesis. BRaf encodes a serine/threonine kinase that regulates the MAPK/ERK kinase (MEK) and extracellular signal-regulated kinase (ERK) protein kinase cascade. In normal cells, the activity of BRaf is tightly regulated and is required for cell growth and survival. BRaf gain-of-function mutations in melanoma frequently lead to unrestrained growth, enhanced cell invasion and increased viability of cancer cells. Although it is clear that the invasive phenotypes of BRaf mutated melanoma cells are stringently dependent on BRaf-MEK-ERK activation, the downstream effector targets that are required for oncogenic BRaf-mediated melanomagenesis are not well defined. miRNAs have regulatory functions towards the expression of genes that are important in carcinogenesis. We observed that miR-10b expression correlates with the presence of the oncogenic BRaf (BRafV600E) mutation in melanoma cells. While expression of miR-10b enhances anchorage-independent growth of BRaf wild-type melanoma cells, miR-10b silencing decreases BRafV600E cancer cell invasion in vitro. Importantly, the expression of miR-10b is required for BRafV600E-mediated anchorage-independent growth and invasion of melanoma cells in vitro. Taken together our results suggest that miR-10b is an important mediator of oncogenic BRafV600E activity in melanoma.

Recent high-throughput-sequencing of cancer genomes has identified oncogenic mutations in the B-Raf genetic locus as one of the critical events in melanomagenesis. B-Raf encodes a serine/threonine kinase that regulates the MAPK/ERK kinase (MEK) and extracellular signal-regulated kinase (ERK) protein kinase cascade. In normal cells, the activity of B-Raf is tightly regulated and is required for cell growth and survival. B-Raf gain-of-function mutations in melanoma frequently lead to unrestrained growth, enhanced cell invasion and increased viability of cancer cells. Although it is clear that the invasive phenotypes of B-Raf mutated melanoma cells are stringently dependent on B-Raf-MEK-ERK activation, the downstream effector targets that are required for oncogenic B-Raf-mediated melanomagenesis are not well defined. miRNAs have regulatory functions towards the expression of genes that are important in carcinogenesis. We observed that miR-10b expression correlates with the presence of the oncogenic B-Raf (B-RafV600E) mutation in melanoma cells. While expression of miR-10b enhances anchorage-independent growth of B-Raf wild-type melanoma cells, miR-10b silencing decreases B-RafV600E cancer cell invasion in vitro. Importantly, the expression of miR-10b is required for B-RafV600E-mediated anchorage independent growth and invasion of melanoma cells in vitro. Taken together our results suggest that miR-10b is an important mediator of oncogenic B-RafV600E activity in melanoma.

Accumulating evidence suggests that presence of macrophages in the tumor microenvironment add to the invasive and tumor-promoting hallmarks of cancer cells by secreting angiogenic and growth factors. RKIP is a known metastasis suppressor and interferes with several steps of metastasis. However, the mechanistic underpinnings of its function as a broad metastasis suppressor remain poorly understood. Here, we establish a novel pathway for RKIP regulation of metastasis inhibition through the negative regulation of RANTES/CCL5 thereby limiting tumor macrophage infiltration and inhibition of angiogenesis. Using a combination of loss- and gain-of- function approaches, we show that RKIP hinders breast cancer cell invasion by inhibiting expression of the CC chemokine CCL5 in vitro. We also show that the expression levels of RKIP and CCL5 are inversely correlated among clinical human breast cancer samples. Using a mouse allograft breast cancer transplantation model, we highlight that ectopic expression of RKIP significantly decreases tumor vasculature, macrophage infiltration and lung metastases. Mechanistically, we demonstrate that the inhibition of the CCL5 expression is the cause of the observed effects resulting from RKIP expression. Taken together, our results underscore the significance of RKIP as important negative regulator of tumor microenvironment.

Recent high-throughput-sequencing of the cancer genome has identified oncogenic mutations inBRafgenetic locus as one of the critical events in melanomagenesis. In normal cells, the activity ofBRafis tightly regulated. Gain-of-function mutations like those identified in melanoma frequently lead to enhanced cell-survival and unrestrained growth. The activating mutation ofBRafwill also induce the cells to senesce. However, the mechanism by which the oncogenicBRafinduces the senescent barrier remains poorly defined. microRNAs have regulatory functions toward the expression of genes that are important in carcinogenesis. Here we show that expression of several microRNAs is altered when the oncogenic version ofBRafis introduced in cultured primary melanocytes and these cells undergo premature cellular senescence. These include eight microRNAs whose expression rates are significantly stimulated and three that are repressed. While most of the induced microRNAs have documented negative effects on cell cycle progression, one of the repressed microRNAs has proven oncogenic functions. Ectopic expression of some of these induced microRNAs increased the expression of senescence markers and induced growth arrest and senescence in primary melanocytes. Taken together, our results suggest that the change in microRNA expression rates may play a vital role in senescence induced by the oncogenicBRaf.

Background: Tumor metastasis suppressors are proteins that impede secondary tumor formation by inhibiting one or more steps of the metastasis cascade without stimulating primary tumor growth. Raf-1 kinase inhibitor protein (RKIP) is a metastasis suppressor that inhibits metastasis in breast, prostate, melanoma and several other types of cancers. The molecular mechanism through which RKIP executes its anti-metastasis effects is not yet completely defined. The objective of the current study is to understand how RKIP inhibits breast cancer cell invasion and metastasis in molecular level. Given its primary function in regulating actin cytoskeleton and cell movements, Rho GTPases were studied as possible downstream effectors of RKIP. These small GTPases belong to the Ras superfamily and consist of nearly 22 members with RhoA and Rac1 as the major players affecting cell motility. Rho GTPases oscillate between a GTP-bound active form and a GDP-bound inactive form. The GDP-GTP cycle is regulated by GEFs that facilitate the exchange of bound GDP with GTP, and by GAPs that stimulate the Rho GTPase activity. Aberrantly activated GEFs are reported to drive tumorigenesis, while activated GAPs are assumed to inhibit the cancer formation and metastasis. Hence, Rho GTPases are widely accepted as oncogenes. Contrary to this notion, several recent studies suggested a possible metastasis inhibitory function for wild-type RhoA in triple negative breast cancer and several other types of cancers. Experimental procedures and results: In this study, several mouse (4T1, 168 FARN) and human (BT20, MDA-MB 231) breast cancer cell lines were used to eliminate possible cell line specific observations. Using in vitro matrigel invasion assay, we showed that RhoA, not Rac1, acts downstream of RKIP and is needed for RKIP-mediated inhibition of breast cancer cell invasion in vitro. GTPase activity pull-down assay results revealed that RKIP specifically increases RhoA activation, thus inhibiting invasiveness of these cells. Similarly, orthotopic mice tumor implantation experiments showed that ectopic expression of dominant negative RhoA in 4T1 cells confers significantly greater proclivity to metastasize in mice. These observations are consistent with RhoA's emerging role as a metastasis suppressor. In BT20 breast cancer cells, RhoA enhanced E-cadherin expression and negatively affected the cell invasiveness. Interestingly, RKIP phenocopied the RhoA effect on E-cadherin and cell invasion, suggesting that RKIP-RhoA axis inhibits breast cancer cell invasiveness by increasing E-cadherin expression. Conclusions: Our results conclude that RKIP specifically increases RhoA activation in breast cancer cells, and this activated RhoA stabilizes E-cadherin and negatively affects the invasiveness of these cells. Citation Format: Gardiyawasam Kalpana, Vu Bach, Clariza Borile, Miranda Yeung, Rafael Garcia-Mata, Kam C. Yeung. RKIP-RhoA axis inhibits breast cancer invasion and metastasis by increasing E-cadherin expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5153.

Purpose External beam radiotherapy (RT) is often used in an attempt to cure localized prostate cancer (PCa), but it is only palliative against disseminated disease. Raf kinase inhibitory protein (RKIP) is a metastasis suppressor whose expression is reduced in approximately 50% of localized PCa tissues and is absent in metastases. Chemotherapeutic agents have been shown to induce tumor apoptosis through induction of RKIP expression. Our goal was to test whether RT similarly induces apoptosis through induction of RKIP expression. Methods and Materials The C4-2B PCa cell line was engineered to overexpress or underexpress RKIP. The engineered cells were tested for apoptosis in cell culture and tumor regression in mice after RT. Results RT induced both RKIP expression and apoptosis of PCa cells. Overexpression of RKIP sensitized PCa cells to radiation-induced apoptosis. In contrast, short-hairpin targeting of RKIP, so that RT could not induce RKIP expression, protected cells from radiation-induced apoptosis. In a murine model, knockdown of RKIP in PCa cells diminished radiation-induced apoptosis. Molecular concept mapping of genes altered on manipulation of RKIP expression revealed an inverse correlation with the concept of genes altered by RT. Conclusion The data presented in this report indicate that the loss of RKIP, as seen in primary PCa tumors and metastases, confers protection against radiation-induced apoptosis. Therefore, it is conceivable that the loss of RKIP confers a growth advantage on PCa cells at distant sites, because the loss of RKIP would decrease apoptosis, favoring proliferation.

Diminished expression of the metastasis suppressor protein RKIP was previously reported in a number of cancers. The underlying mechanism remains unknown. Here, we show that the expression of RKIP negatively correlates with that of Snail zinc-transcriptional repressor, a key modulator of normal and neoplastic epithelial-mesenchymal transition (EMT) program. With a combination of loss-of-function and gain-of-function approaches, we showed that Snail repressed the expression of RKIP in metastatic prostate cancer cell lines. The effect of Snail on RKIP was on the level of transcriptional initiation and mediated by a proximal E-box on the RKIP promoter. Our results therefore suggest that RKIP is a novel component of the Snail transcriptional regulatory network important for the progression and metastasis of cancer.

Raf-1 kinase inhibitor protein (RKIP) has been implicated in the regulation of cell survival pathways and metastases, and is poorly expressed in tumors. We have reported that the NF-κB pathway regulates tumor resistance to apoptosis by the TNF-α family via inactivation of the transcription repressor Yin Yang 1 (YY1). We hypothesized that RKIP overexpression may regulate tumor sensitivity to death ligands via inhibition of YY1 and up-regulation of death receptors (DRs). The TRAIL-resistant prostate carcinoma PC-3 and melanoma M202 cell lines were examined. Transfection with CMV-RKIP, but not with control CMV-EV, sensitized the cells to TRAIL-mediated apoptosis. Treatment with RKIP small interfering RNA (siRNA) inhibited TRAIL-induced apoptosis. RKIP overexpression was paralleled with up-regulation of DR5 transcription and expression; no change in DR4, decoy receptor 1, and decoy receptor 2 expression; and inhibition of YY1 transcription and expression. Inhibition of YY1 by YY1 siRNA sensitized the cells to TRAIL apoptosis concomitantly with DR5 up-regulation. RKIP overexpression inhibited several antiapoptotic gene products such as X-linked inhibitor of apoptosis (XIAP), c-FLIP long, and Bcl-xL that were accompanied with mitochondrial membrane depolarization. RKIP overexpression in combination with TRAIL resulted in the potentiation of these above effects and activation of caspases 8, 9, and 3, resulting in apoptosis. These findings demonstrate that RKIP overexpression regulates tumor cell sensitivity to TRAIL via inhibition of YY1, up-regulation of DR5, and modulation of apoptotic pathways. We suggest that RKIP may serve as an immune surveillance cancer gene, and its low expression or absence in tumors allows the tumor to escape host immune cytotoxic effector cells.

Raf kinase inhibitory protein (RKIP-1) is involved in the regulation of the MAP kinase, NF-kappaB, and GPCR signaling pathways. It is expressed in numerous tissues and cell types and orthologues have been documented throughout the animal and plant kingdoms. RKIP-1 has also been reported as an inhibitor of serine proteases, and a precursor of a neurostimulatory peptide. RKIP-1 has been implicated as a suppressor of metastases in several human cancers. We generated a knockout strain of mice to further assess RKIP-1's function in mammals. RKIP-1 is expressed in many tissues with the highest protein levels detectable in testes and brain. In the brain, expression was ubiquitous in limbic formations, and homozygous mice developed olfaction deficits in the first year of life. We postulate that RKIP-1 may be a modulator of behavioral responses.

Raf Kinase Inhibitory Protein or RKIP was initially identified as a Raf-1 binding protein using the yeast 2-hybrid screen. RKIP inhibits the activation phosphorylation of MEK by Raf-1 by competitively inhibiting the binding of MEK to Raf-1 and thus exerting an inhibitory effect on the Raf-MEK-Erk pathway. RKIP has been identified as a metastasis suppressor gene. Expression of RKIP is low in cancer metastases. Although primary tumor growth remains unaffected, re- expression of RKIP inhibits cancer metastasis. Mechanistically, RKIP constrains metastasis by inhibiting angiogenesis, local invasion, intravasation, and colonization. The molecular mechanism of how RKIP inhibits these individual steps remains undefined. In our present study, using an unbiased PCR based screening and by analyzing DNA microarray expression datasets we observe that the expression of multiple metalloproteases (MMPs) including MMP1, MMP3, MMP10 and MMP13 are negatively correlated with RKIP expression in breast cancer cell lines and clinical samples. Since expression of MMPs by cancer cells is important for cancer metastasis, we hypothesize that RKIP may mediate suppression of breast cancer metastasis by inhibiting multiple MMPs. We show that the expression signature of RKIP and MMPs is better at predicting high metastatic risk than the individual gene. Using a combination of loss- and gain-of-function approaches, we find that MMP13 is the cause of RKIP-mediated inhibition of local cancer invasion. Interestingly expression of MMP13 alone is not sufficient to reverse the inhibition of breast cancer cell metastasis to the lung due to the expression of RKIP. We find that RKIP negatively regulates MMP13 through the Erk2 signaling pathway and the repression of MMP13 by RKIP is transcription factor AP-1 independent. Together, our findings indicate that RKIP inhibits cancer cell invasion, in part, via MMP13 inhibition. These data also implicate RKIP in the regulation of MMP transcription, suggesting a potential mechanism by which RKIP inhibits tumor progression and metastasis.

The Raf-MEK-ERK protein kinase cascade is a highly conserved signaling pathway that is pivotal in relaying environmental cues from the cell surface to the nucleus. Three Raf isoforms, which share great sequence and structure similarities, have been identified in mammalian cells. We have previously identified Raf kinase inhibitor protein (RKIP) as a negative regulator of the Raf-MEK-ERK signaling pathway by specifically binding to the Raf-1 isoform. We show here that RKIP also antagonizes kinase activity of the B-Raf isoform. Yeast two-hybrid and coimmunoprecipitation experiments indicated that RKIP specifically interacted with B-Raf. Ectopic expression of RKIP antagonized the kinase activity of B-Raf. We showed that the effects of RKIP on B-Raf functions were independent of its known inhibitory action on Raf-1. The expression levels of RKIP in melanoma cancer cell lines are low relative to primary melanocytes. Forced expression of RKIP partially reverted the oncogenic B-Raf kinase-transformed melanoma cancer cell line SK-Mel-28. The low expression of RKIP and its antagonistic action on B-Raf suggests that RKIP may play an important role in melanoma turmorgenesis.

Cancer cells are more susceptible to chemotherapeutic agent-induced apoptosis than their normal counterparts. Although it has been demonstrated that the increased sensitivity results from deregulation of oncoproteins during cancer development (Evan, G. I., and Vousden, K. H. (2001) Nature 411, 342-348; Green, D. R., and Evan, G. I. (2002) Cancer Cell 1, 19-30), little is known about the signaling pathways leading to changes in the apoptotic threshold in cancer cells. Here we show that low RKIP expression levels in tumorigenic human prostate and breast cancer cells are rapidly induced upon chemotherapeutic drug treatment, sensitizing the cells to apoptosis. We show that the maximal RKIP expression correlates perfectly with the onset of apoptosis. In cancer cells resistant to DNA-damaging agents, treatment with the drugs does not up-regulate RKIP expression. However, ectopic expression of RKIP resensitizes DNA-damaging agent-resistant cells to undergo apoptosis. This sensitization can be reversed by up-regulation of survival pathways. Down-regulation of endogenous RKIP by expression of antisense and small interfering RNA (siRNA) confers resistance on sensitive cancer cells to anticancer drug-induced apoptosis. Our studies suggest that RKIP may represent a novel effector of signal transduction pathways leading to apoptosis and a prognostic marker of the pathogenesis of human cancer cells and tumors after treatment with clinically relevant chemotherapeutic drugs.

One key area of protein kinase research is the identification of cognate substrates. The search for substrates is hampered by problems in unambiguously assigning substrates to a particular kinase in vitro and in vivo. One solution to this impasse is to engineer the kinase of interest to accept an ATP analogue which is orthogonal (unable to fit into the ATP binding site) for the wild-type enzyme and the majority of other kinases. The acceptance of structurally modified, gamma-(32)P-labelled, nucleotide analogue by active site-modified kinase can provide a unique handle by which the direct substrates of any particular kinase can be displayed in crude mixtures or cell lysates. We have taken this approach with the serine/threonine kinase Raf-1, which plays an essential role in the transduction of stimuli through the Ras-->Raf-->MEK-->ERK/MAP kinase cascade. This cascade plays essential roles in proliferation, differentiation and apoptosis. Here we detail the mutagenesis strategy for the ATP binding pocket of Raf-1, such that it can utilise an N(6)-substituted ATP analogue. We show that these mutations do not alter the substrate specificity and signal transduction through Raf-1. We screen a library of analogues to identify which are orthogonal for Raf-1, and show that mutant Raf-1 can utilise the orthogonal analogue N(6)(2-phenethyl) ATP in vitro to phosphorylate its currently only accepted substrate MEK. Importantly we show that our approach can be used to tag putative direct substrates of Raf-1 kinase with (32)P-N(6)(2-phenethyl) ATP in cell lysates.

Raf kinase inhibitor protein (RKIP) is a member of the phosphatidylethanolamine-binding-protein (PEBP) family that modulates the action of many kinases involved in cellular growth, apoptosis, epithelial to mesenchymal transition, motility, invasion and metastasis. Previously, we described an inverse association between RKIP and signal transducers and activators of transcription 3 (STAT3) expression in gastric adenocarcinoma patients. In this study, we elucidated the mechanism by which RKIP regulates STAT3 activity in breast and prostate cancer cell lines. RKIP over expression inhibited c-Src auto-phosphorylation and activation, as well as IL-6-, JAK1 and 2-, and activated Raf-mediated STAT3 tyrosine and serine phosphorylation and subsequent activation. In MDA-231 breast cancer cells that stably over express RKIP, IL-6 treatment blocked STAT3 phosphorylation and transcriptional activation. Conversely, in RKIP knockdown MDA-231 cells: STAT3 phosphorylation and activation increased in comparison to parental MDA-231 cells. RKIP over expression resulted in constitutive physical interaction with STAT3 and blocked c-Src and STAT3 association. The treatment of DU145 prostate, but not PC3 prostate or MDA-231 breast, cancer cell lines with ENMD-1198 or MKC-1 dramatically increased expression of RKIP. Overexpression of RKIP sensitized PC3 and MDA-231 cells to MTI-induced apoptosis. Moreover, MTI treatment resulted in a decrease in Src-mediated STAT3 tyrosine phosphorylation and activation, an effect that was significantly enhanced by RKIP over expression. In stable RKIP over expressing MDA-231 cells, tumor xenograft growth induced by activated STAT3 is inhibited. RKIP synergizes with MTIs to induce apoptosis and inhibit STAT3 activation of breast and prostate cancer cells. RKIP plays a critical role in opposing the effects of pro-oncogenic STAT3 activation.

The sources and consequences of nongenetic variability in metastatic progression are largely unknown. To address these questions, we characterized a transcriptional regulatory network for the metastasis suppressor Raf kinase inhibitory protein (RKIP). We previously showed that the transcription factor BACH1 is negatively regulated by RKIP and promotes breast cancer metastasis. Here we demonstrate that BACH1 acts in a double-negative (overall positive) feedback loop to inhibit RKIP transcription in breast cancer cells. BACH1 also negatively regulates its own transcription. Analysis of the BACH1 network reveals the existence of an inverse relationship between BACH1 and RKIP involving both monostable and bistable transitions that can potentially give rise to nongenetic variability. Single-cell analysis confirmed monostable and bistable-like behavior. Treatment with histone deacetylase inhibitors or depletion of the polycomb repressor enhancer of zeste homolog 2 altered relative RKIP and BACH1 levels in a manner consistent with a prometastatic state. Together, our results suggest that the mutually repressive relationship between metastatic regulators such as RKIP and BACH1 can play a key role in determining metastatic progression in cancer.

Raf Kinase inhibitory protein (RKIP) is a well-established metastasis suppressor that is frequently downregulated in aggressive cancers. The impact of RKIP and its phosphorylated form on disease-free survival (DFS) and other clinicopathological parameters in breast cancer is yet to be discovered. To this end, we examined RKIP expression in 3 independent breast cancer cohorts. At the Protein level, loss or reduced total RKIP expression was associated with large-sized tumors characterized by high proliferative index, high-grade and diminished estrogen (ER) and progesterone receptor expression. Loss or diminution of RKIP expression was significantly associated with shorter DFS in all cohorts. Moreover, the complete loss of p-RKIP was an independent prognostic factor using multivariate analysis in operable invasive ductal breast cancer. We show for the first time that ER, partly, drives RKIP expression through MTA3-Snail axis. Consistent with this finding, we found that, at the mRNA level, RKIP expression varied significantly across the different molecular subtypes of breast cancer with the Luminal (ER+) subtype expressing high levels of RKIP and the more aggressive Claudin-low (ER-) subtype, which depicted the highest epithelial to mesenchymal transition (EMT) registered the lowest RKIP expression levels. In conclusion, loss of expression/diminution of RKIP or its phosphorylated form is associated with poor diseases-free survival in breast cancer. Determining the expression of RKIP and p-RKIP adds significant prognostic value to the management and subtyping of this disease.

Background/Aims: Raf kinase inhibitory protein (RKIP) is a scaffolding molecule in the PEBP family that sequesters certain signaling molecules away from their pathways, thereby abrogating intracellular growth signals. RKIP has been assigned multiple functions and is associated with an increasing number of diseases through its involvement with signal transduction pathways. We previously demonstrated that RKIP is highly expressed in human normal prostate epithelial cells and plays a pivotal role during prostate cancer (PCa) progression. Whether RKIP is subject to endocrine regulation has not been reported. Methods: The effect of dihydrotestosterone (DHT) on RKIP expression in normal prostate epithelial cells was determined by real-time RTPCR and Western blot. Report assay was performed to determine whether the regulation of RKIP by androgens is at the transcriptional level. The binding of androgen receptor (AR) to the RKIP promoter was determined by EMSA and Chromatin Immunoprecipitation (ChIP) assays. To determine whether RKIP was regulated by androgen in vivo, we examined RKIP expression level in response to castration in 6-8 week old C57BL/6 male mice. Results: Here we report that DHT positively regulates the transcription of RKIP in the normal prostate epithelial cells. The anti-androgen bicalutamide blocked androgen-mediated regulation of RKIP, which indicates that this regulation is mediated through AR. Transfection of the cells with a RKIP promoter-driven luciferase reporter vector showed that DHT increased RKIP promoter activity in parallel with changes in expression. EMSA demonstrates that AR binds to a putative ARE in the RKIP promoter, which was further validated by ChIP assay. Importantly, these data are further supported by our in vivo experiment where castrated mice had less RKIP expression in their prostate glands than sham-operated mice. Conclusions: Collectively, the results establish RKIP as a novel androgen target gene. Androgens induce RKIP expression through AR-mediated transcriptional modulation of the RKIP promoter in the prostate. This is the first demonstration of endocrine regulation of the metastasis suppressor gene RKIP.

Epigenetic modifications such as histone methylation play an important role in human cancer metastasis. Enhancer of zeste homolog 2 (EZH2), which encodes the histone methyltransferase component of the polycomb repressive complex 2 (PRC2), is overexpressed widely in breast and prostate cancers and epigenetically silences tumor suppressor genes. Expression levels of the novel tumor and metastasis suppressor Raf-1 kinase inhibitor protein (RKIP) have been shown to correlate negatively with those of EZH2 in breast and prostate cell lines as well as in clinical cancer tissues. Here, we show that the RKIP/EZH2 ratio significantly decreases with the severity of disease and is negatively associated with relapse-free survival in breast cancer. Using a combination of loss- and gain-of-function approaches, we found that EZH2 negatively regulated RKIP transcription through repression-associated histone modifications. Direct recruitment of EZH2 and suppressor of zeste 12 (Suz12) to the proximal E-boxes of the RKIP promoter was accompanied by H3-K27-me3 and H3-K9-me3 modifications. The repressing activity of EZH2 on RKIP expression was dependent on histone deacetylase promoter recruitment and was negatively regulated upstream by miR-101. Together, our findings indicate that EZH2 accelerates cancer cell invasion, in part, via RKIP inhibition. These data also implicate EZH2 in the regulation of RKIP transcription, suggesting a potential mechanism by which EZH2 promotes tumor progression and metastasis. Cancer Res; 72(12); 3091–104. ©2012 AACR.

Human TFIIA activity is composed of three subunits (alpha, beta, gamma). Here we report the isolation of a human cDNA clone encoding the gamma-subunit and the reconstitution of TFIIA activity from recombinant polypeptides (holo-TFIIA). Protein-protein interaction analysis established that the beta and gamma subunits of TFIIA interact with the TBP component of TFIID. The alpha-subunit is recruited into the complex by association with the gamma-subunit. Functional studies indicate that recombinant TFIIA stimulates basal TFIID-dependent transcription but is without effect on TBP-dependent transcription. Our studies indicate that TFIIA not only functions by physically removing negative components present in TFIID (antirepression), as demonstrated previously, but that it can stimulate basal transcription through components of the TFIID complex. Holo-TFIIA also stimulated activation of transcription in vitro as well as in vivo in transfected HeLa cells.

The novel proteasome inhibitor NPI-0052 has been shown to sensitize tumor cells to apoptosis by various chemotherapeutic drugs and tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), although the mechanisms involved are not clear. We hypothesized that NPI-0052–mediated sensitization may result from NF-κB inhibition and downstream modulation of the metastasis inducer Snail and the metastasis suppressor/immunosurveillance cancer gene product Raf-1 kinase inhibitory protein (RKIP). Human prostate cancer cell lines were used as models, as they express different levels of these proteins. We show that NPI-0052 inhibits both NF-κB and Snail and induces RKIP expression, thus resulting in cell sensitization to CDDP and TRAIL. The direct role of NF-κB inhibition in sensitization was corroborated with the NF-κB inhibitor DHMEQ, which mimicked NPI-0052 in sensitization and inhibition of Snail and induction of RKIP. The direct role of Snail inhibition by NPI-0052 in sensitization was shown with Snail small interfering RNA, which reversed resistance and induced RKIP. Likewise, the direct role of RKIP induction in sensitization was revealed by both overexpression of RKIP (mimicking NPI-0052) and RKIP small interfering RNA that inhibited NPI-0052–mediated sensitization. These findings show that NPI-0052 modifies the NF-κB-Snail-RKIP circuitry in tumor cells and results in downstream inhibition of antiapoptotic gene products and chemoimmunosensitization. The findings also identified Snail and RKIP as targets for reversal of resistance. [Cancer Res 2009;69(21):8376–85]

Raf Kinase Inhibitory Protein or RKIP was initially identified as a Raf-1 binding protein using the yeast 2-hybrid screen. RKIP inhibits the activation phosphorylation of MEK by Raf-1 by competitively inhibiting the binding of MEK to Raf-1 and thus exerting an inhibitory effect on the Raf-MEK-ERK pathway. RKIP has been identified as a metastasis suppressor gene. Metastasis is a complex multiple-step process. Expression of RKIP is low in cancer metastases. Although primary tumor growth was unaffected, re-expression of RKIP inhibits cancer metastasis. Mechanistically, RKIP constrains metastasis by inhibiting angiogenesis, local invasion, intravasation, and colonization. The molecular mechanism of how RKIP inhibits these individual steps remains undefined. Using an unbiased PCR based screening and by analyzing DNA microarray expression datasets we observed that the expression of multiple metalloproteases (MMP) including MMP1, MMP3, MMP10 and MMP13 were negatively correlated with RKIP expression in breast cancer cell lines and clinical samples. Since expression of MMPs by cancer cells is important for cancer metastasis, we hypothesize that RKIP may mediate suppression of breast cancer metastasis by inhibiting multiple MMPs. In this study we show that the expression signature of RKIP and MMPs is better at predicting high metastatic risk than the individual gene. Using a combination of loss- and gain-of-function approaches, we found that MMP13 is the cause of RKIP-mediated inhibition of local cancer invasion. Interestingly expression of MMP13 alone was not sufficient to reverse the inhibition of breast cancer cells metastasis to the lung due to the expression of RKIP. We found that RKIP negatively regulated MMP13 through the Erk2-Fra1 transcriptional axis and the repression of MMP13 by RKIP requires the presence of the a TPA-responsive element in the MMP13 proximal promoter. Together, our findings indicate that RKIP inhibits cancer cell invasion, in part, via MMP13 inhibition. These data also implicate RKIP in the regulation of MMPs transcription, suggesting a potential mechanism by which RKIP inhibits tumor progression and metastasis. Citation Format: Ila J. Datar, Jingwei Feng, Gang Ren, Zehui Li, Xiaoliang Qiu, Fahd Al-Mulla, Milad S. Bitar, Miranda Yeung, Kam Yeung. Effect of RKIP on MMP expression and regulation of breast cancer metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 124. doi:10.1158/1538-7445.AM2014-124

The Raf kinase inhibitor protein (RKIP) acts as a negative regulator of the mitogen-activated protein (MAP) kinase (MAPK) cascade initiated by Raf-1. RKIP inhibits the phosphorylation of MAP/extracellular signal-regulated kinase 1 (MEK1) by Raf-1 by disrupting the interaction between these two kinases. We show here that RKIP also antagonizes the signal transduction pathways that mediate the activation of the transcription factor nuclear factor kappa B (NF-kappaB) in response to stimulation with tumor necrosis factor alpha (TNF-alpha) or interleukin 1 beta. Modulation of RKIP expression levels affected NF-kappaB signaling independent of the MAPK pathway. Genetic epistasis analysis involving the ectopic expression of kinases acting in the NF-kappaB pathway indicated that RKIP acts upstream of the kinase complex that mediates the phosphorylation and inactivation of the inhibitor of NF-kappaB (IkappaB). In vitro kinase assays showed that RKIP antagonizes the activation of the IkappaB kinase (IKK) activity elicited by TNF-alpha. RKIP physically interacted with four kinases of the NF-kappaB activation pathway, NF-kappaB-inducing kinase, transforming growth factor beta-activated kinase 1, IKKalpha, and IKKbeta. This mode of action bears striking similarities to the interactions of RKIP with Raf-1 and MEK1 in the MAPK pathway. Emerging data from diverse organisms suggest that RKIP and RKIP-related proteins represent a new and evolutionarily highly conserved family of protein kinase regulators. Since the MAPK and NF-kappaB pathways have physiologically distinct roles, the function of RKIP may be, in part, to coordinate the regulation of these pathways.

Raf-1 phosphorylates and activates MEK-1, a kinase that activates the extracellular signal regulated kinases (ERK). This kinase cascade controls the proliferation and differentiation of different cell types. Here we describe a Raf-1-interacting protein, isolated using a yeast two-hybrid screen. This protein inhibits the phosphorylation and activation of MEK by Raf-1 and is designated RKIP (Raf kinase inhibitor protein). In vitro, RKIP binds to Raf-1, MEK and ERK, but not to Ras. RKIP co-immunoprecipitates with Raf-1 and MEK from cell lysates and colocalizes with Raf-1 when examined by confocal microscopy. RKIP is not a substrate for Raf-1 or MEK, but competitively disrupts the interaction between these kinases. RKIP overexpression interferes with the activation of MEK and ERK, induction of AP-1-dependent reporter genes and transformation elicited by an oncogenically activated Raf-1 kinase. Downregulation of endogenous RKIP by expression of antisense RNA or antibody microinjection induces the activation of MEK-, ERK- and AP-1-dependent transcription. RKIP represents a new class of protein-kinase-inhibitor protein that regulates the activity of the Raf/MEK/ERK module.

The heterotetrameric Dr1-DRAP1 transcriptional repressor complex was functionally dissected. Dr1 was found to contain two domains required for repression of transcription. The tethering domain interacts with the TATA box binding protein and directs the repressor complex to the promoter. This tethering domain can be replaced by a domain conferring sequence-specific recognition to the repressor complex. In the absence of the tethering domain, Dr1 interacts with its corepressor DRAP1, but this interaction is not functional. The enhancement of Dr1-mediated repression of transcription by DRAP1 requires the tethering domain. The second domain of Dr1 is the repression domain, which is glutamine-alanine rich. A 65-amino-acid polypeptide containing the repression domain fused to the Ga14 DNA binding domain repressed transcription when directed to TATA-containing and TATA-less promoters. This repression domain was also found to functionally and directly interact with the TATA box binding protein.

The sources of nongenetic variability in metastatic progression are largely unknown. To address this question, we characterized a transcriptional regulatory network for the metastasis suppressor Raf Kinase Inhibitory Protein (RKIP). We previously showed that the transcription factor BACH1 is negatively regulated by RKIP, promotes breast cancer metastasis, and negatively correlates with RKIP expression in primary breast tumors. Here we demonstrate that BACH1 acts in a double negative (overall positive) feedback loop to inhibit RKIP transcription in breast cancer cells. BACH1 also negatively regulates its own transcription. HDAC inhibitors increased BACH1 but not RKIP transcription, whereas depletion of polycomb repressor EZH2 induced RKIP transcription. Analysis of the BACH1 network reveals the existence of a bistable, inverse relationship between BACH1 and RKIP that can potentially give rise to nongenetic variability. Together, our results suggest that the balance between RKIP and BACH1 plays a key role in determining metastatic progression in breast cancers. Citation Format: Jiyoung Lee, Jieun Yun, Kam Yeung, Elena Bevilacqua, Gábor Balázsi, Marsha Rich Rosner. BACH1 and RKIP participate in a bistable network that affects progression to metastasis in breast Cancer. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A39.

Breast cancer mortality is primarily due to metastasis to organs including bone, lungs, liver and brain. Tumor metastasis suppressors are metastasis inhibitors but do not affect the ability of the transformed cells to produce a tumor at the primary sites. Raf kinase inhibitory protein (RKIP) has been identified as a metastasis suppressor for a number of cancers including breast, prostate, liver, colon, skin, and nasopharyngeal carcinoma. RKIP overexpression in hepatoma, melanoma, and prostate metastatic cell lines diminished their invasiveness and motility in vitro. The molecular mechanism by which RKIP transcription is regulated in breast cancer is largely unknown. Interestingly, RKIP expression was highly and positively correlated with patient's metastasis-free survival after radiotherapy. Likewise, when RKIP was reduced in human prostate cancer cells, radiation-induced apoptosis was reduced in xenograft tumors. Moreover, RKIP expression levels have been induced upon chemotherapeutic drug treatment in human breast cancer cells, sensitizing the tumor cells to apoptosis. These studies indicate that chemotherapy and radiotherapy are more effective when RKIP is present in the metastatic cancer cells. Therefore, high RKIP levels are desirable to prevent metastasis of breast tumor cells and to sensitize tumor cells for chemotherapy. Our goal is to understand the mechanism by which RKIP is regulated in order to restore its levels. Previously, we have observed that let-7 negatively regulates two target genes, BACH1 and HMGA2 that promote breast cancer metastasis in a xenograft mouse model. Here we demonstrate that BACH1 and HMGA2 are themselves negative regulators of RKIP. Specifically, we have delineated molecular mechanisms by which BACH1 and HMGA2 inhibit RKIP expression in breast cancer cell lines using both luciferase reporter assays and chromatin immunoprecipitation (ChIP) assays. We are also analyzing whether BACH1 and HMGA2 are dysregulated by chemotherapy in highly metastatic breast cancer cells and whether suppression of these RKIP regulators can potentiate sensitivity of the breast cancer cells to chemotherapy. These studies provide valuable mechanistic insights for potentiating chemotherapeutic sensitivity as well as potential biomarkers for metastatic breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3433. doi:1538-7445.AM2012-3433

Therapeutic resistance remains the most challenging aspect of treating cancer. Raf kinase inhibitory protein (RKIP) emerged as a molecule capable of sensitizing cancerous cells to radio- and chemotherapy. Moreover, this small evolutionary conserved molecule, endows significant resistance to cancer therapy when its expression is reduced or lost. RKIP has been shown to inhibit the Raf-MEK-ERK, NFκB, GRK and activate the GSK3β signaling pathways. Inhibition of Raf-MEK-ERK and NFκB remains the most prominent pathways implicated in the sensitization of cells to therapeutic drugs. Our purpose was to identify a possible link between RKIP-KEAP 1-NRF2 and drug resistance. To that end, RKIP-KEAP 1 association was tested in human colorectal cancer tissues using immunohistochemistry. RKIP miRNA silencing and its inducible overexpression were employed in HEK-293 immortalized cells, HT29 and HCT116 colon cancer cell lines to further investigate our aim. We show that RKIP enhanced Kelch-like ECH-associated protein1 (KEAP 1) stability in colorectal cancer tissues and HT29 CRC cell line. RKIP silencing in immortalized HEK-293 cells (termed HEK-499) correlated significantly with KEAP 1 protein degradation and subsequent NRF2 addiction in these cells. Moreover, RKIP depletion in HEK-499, compared to control cells, bestowed resistance to supra physiological levels of H(2)O(2) and Cisplatin possibly by upregulating NF-E2-related nuclear factor 2 (NRF2) responsive genes. Similarly, we observed a direct correlation between the extent of apoptosis, after treatment with Adriamycin, and the expression levels of RKIP/KEAP 1 in HT29 but not in HCT116 CRC cells. Our data illuminate, for the first time, the NRF2-KEAP 1 pathway as a possible target for personalized therapeutic intervention in RKIP depleted cancers.

Dr1, a repressor of class II genes, regulates transcription by a novel mechanism. Biochemical analyses reveal that Dr1 directly interacts with the multiprotein TFIID complex. By use of the yeast two-hybrid system, we demonstrate that the association of Dr1 with the TATA-binding protein (TBP) subunit of TFIID occurs in vivo. In addition, Dr1 can repress transcription from TATA-containing as well as TATA-less promoters in transient transfection assays. Importantly, Dr1-mediated repression can be reversed by overexpression of TBP in vivo. By use of diverse approaches, we mapped two distinct domains in Dr1 required for repression. One domain is essential for the Dr1-TBP interaction, and the second is rich in alanine residues. The TBP-binding domain of Dr1 cannot be replaced by a heterologous DNA-binding domain in mediating repression. We demonstrate that some, but not all, transcriptional activators can reverse Dr1-mediated repression in vivo.

Past experiments have shown that the adenovirus E1A12S product activates the hsp70 promoter, dependent on the TATA element and dependent on N-terminal E1A sequences. Other experiments have identified a factor termed Dr1 that interacts with and inhibits the transcriptional activity of the TATA-binding protein (TBP). We now find that the E1A12S protein can disrupt the interaction of the Dr1 factor with the TATA-specific TBP factor, allowing the productive interaction of TBP with TFIIA. This E1A-mediated disruption is dependent on N-terminal sequences that are also essential for the TATA-dependent trans-activation of the hsp70 promoter. Moreover, we also find that Dr1 expression in transfected cells can inhibit transcription from the hsp70 promoter and that this can be overcome by coexpression of the wild-type E1A protein, dependent on N-terminal sequences. We conclude that the activation of hsp70 through the TATA element may be mechanistically similar to the activation of the E2 promoter via E2F, in each case involving a release of a transcription factor from an inactive complex.

The Raf kinase inhibitory protein (RKIP) acts as an activator of apoptosis and inhibitor of metastasis in prostate cancer. Diminished expression of RKIP has been reported in several cancers including prostate carcinomas; however, the underlying mechanism of RKIP regulation remains largely unknown. RKIP transcription is inhibited by the EMT inducer Snail which also represses the expression of the metastasis suppressor E-cadherin. E-cadherin and RKIP are co-expressed in many tumors. E-cadherin transcription is also suppressed by the polycomp repressive complex (PRP) transcription repressor, enhancer of zeste homolog 2 (EZH2), which mediates an H3 lysine 27 trimethylation in target promoters. EZH2 is highly expressed in several metastatic tumors including prostate cancer and its levels are inversely correlated with E-cadherin and RKIP expressions. The zinc-finger transcription factor Yin-Yang 1 (YY1) is also overexpressed in prostate tumors and has been associated with tumor progression. Silencing of YY1 by siRNA reverses the EMT phenotype of metastatic prostate cancer cell lines by induction of E-cadherin and RKIP. Here, we explored the direct roles of YY1 and EZH2 in the transcriptional regulation of RKIP in prostate cancer. We used as experimental models the metastatic and non-metastatic prostate cancer cell lines DU145 and LNCaP, respectively, which differ in the expression levels of EZH2, YY1 and RKIP. DU145 cells express low RKIP and high EZH2 and YY1 levels, whereas LNCaP cells express the opposite. Putative binding sites were identified for both YY1 and EZH2 (proximal E-boxes) within the RKIP promoter. CHiP analyses showed that both factors can physically associate with the RKIP promoter. Loss and gain of function reporter approaches showed that ectopic expression of EZH2 in LNCaP cells inhibits the wild type, but not the E-box mutant RKIP basal promoter activity. Similarly, a mutated EZH2 vector that lacks efficient binding to the target promoter couldn't inhibit the RKIP promoter activity. EZH2-mediated inhibition of RKIP promoter activity could also be inhibited by HDAC inhibitors, suggesting that HDAC recruitment and deacetylation of histone H3 is perquisite for EZH2 suppressive activity. Silencing of EZH2 or YY1 in DU145 also resulted in induction of RKIP promoter activity. The negative effect of YY1 on RKIP promoter activity was corroborated by inhibition of RKIP mRNA and protein levels. The above observations were further validated in prostate cancer tissue microarrays whereby YY1 and RKIP protein expressions were found to be inversely correlated. The present findings support the roles of EZH2 and YY1 as transcription repressors of RKIP in prostate cancer and suggest that their overexpression may be associated, in part, with the high cell metastatic potential through downregulation of RKIP. We propose that EZH2 and YY1 might be potential prognostic markers of prostate cancer progression and targets for therapeutic intervention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2162. doi:10.1158/1538-7445.AM2011-2162

The Raf Kinase Inhibitor Protein (RKIP) is a member of the phosphatidylethanolamine binding protein family (PEBP). RKIP binds to Raf-1 and prevents the activation of the ERK 1/2 cascade. RKIP expression is diminished in many primary cancers and is absent in many metastases. RKIP overexpression inhibits experimental metastases, hence, termed a metastasis suppressor. In addition, overexpression of RKIP reverses tumor cell resistance to drug-induced apoptosis, hence, termed an immune surveillance cancer gene product. Phosphorylation of RKIP (pRKIP) at Ser 153 by PKCs dissociates RKIP from Raf-1 (inactivating RKIP) and pRKIP binds to G-protein coupled receptor kinase 2 (GRK2, a kinase that inhibits G-protein coupled receptors (GPCRs) and resulting in the inhibition of GRK2 activity and stimulation of the ERK 1/2 activity. Over 90% of melanoma express oncogene mutations in BRAF and also express mutations in NRAS. Most of the transforming activity of these mutations results from the activation of MAPK. The roles of such mutations in the regulation of oncogenes involved in metastasis and resistance are not clear. The objective of this study was to initiate investigations on the roles of above mutations on the expression of RKIP and inactive pRKIP. We hypothesized that BRAF and NRAS mutations, through their activation of PKCs, will result in the phosphorylation and inactivation of RKIP and potentiation of ERK 1/2 activation, metastasis and drug resistance. In this study, we have examined a large panel of melanoma cell lines with no mutations and with BRAF and/or NRAS mutations using a Tissue microarray technology. The expression of both RKIP and pRKIP was examined and the evaluation of protein expression was performed by IHC and analyzed by blinded observers under light microscopy. While anti-RKIP antibody detects both RKIP and pRKIP, anti-pRKIP was specific for pRKIP. The mean intensities for the expression of each protein were determined using the software Image-ProPlus 6.3. In brief, five random fields of each cell line were evaluated at 400-magnification. From the IHC staining, general density expressions of each protein in the cell lines were measured in pixels/200 μm2. The data revealed that the majority (75%) of RKIP expression was in its phosphorylated form in cell lines with BRAF and NRAS mutations. These studies suggest that in melanoma cell lines with mutations, GRK2 is inactivated through its association with pRKIP and, thus, maintaining the activating signals mediated by GPCRs. Further, pRKIP expression may potentiate the metastatic potential and drug resistance. We propose that selective inhibitors of PKCs may elevate the expression of RKIP and result in the inhibition of the metastatic potential as well as in the sensitization of the tumor cells to subtoxic therapeutic drugs. These studies are currently being examined in our laboratories. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3374.

Nitric oxide (NO) has been shown to mediate contrasting effects on tumorigenesis i.e. at low levels NO is pro-tumorigenic and at high levels NO is anti-tumorigenic. Recent studies revealed that treatment of tumor cell lines with NO donors at high levels significantly inhibited cell growth and survival pathways such as the constitutively activated NF-κB. Tumor cells with activated NF-κB were reported to have the capacity to initiate the metastatic cascade via induction of epithelial to mesenchymal transition (EMT). The EMT phenotype is characterized by decreased expression of epithelial gene products (e.g. E-cadherin, cytokeratin 18) and upregulation of mesenchymal gene products (e.g. vimentin, fibronectin). In addition, the tumor cells exhibit invasive and migratory properties. Based on these above findings, we hypothesized that treatment of metastatic prostate carcinoma cell lines, PC-3 and DU-145 with DETANONOate (500-1000μM) may reverse the EMT phenotype. This hypothesis was tested and treatment of the cells with DETANONOate resulted in inhibition of the EMT phenotype through inhibition of NF-κB activity. Inhibition of NF-κB was shown to be due, in part, to S-nitrosylation of p50. In addition to the inhibition of EMT phenotype, treatment with DETANONOate also inhibited invasive properties. The dual roles of DETANONOate-mediated inhibition of NF-κB and EMT were corroborated by the use of the NF-κB inhibitor DHMEQ. We examined the underlying mechanism by which NF-κB-induced inhibition by DETANONOate resulted in the inhibition of EMT. The EMT-induced transcription factor Snail was highly expressed in PC-3 and DU-145 cells. Treatment with DETANONOate inhibited Snail expression concomitant with inhibition of EMT. The direct role of Snail inhibition by DETANONOate and the inhibition of EMT was corroborated in cells transfected with Snail siRNA and such treatment reversed the EMT phenotype, mimicking DETANONOate. Inhibition of the repressor activity of Snail by DETANONOate resulted in the upregulation of the Raf-1 kinase inhibitory protein (RKIP). The role of RKIP induction in the reversal of EMT was corroborated in cells overexpressing RKIP. The in vitro findings with DETANONOate-induced inhibition of EMT were validated in mice bearing PC-3 xenografts. Treatment with DETANONOate and analysis by IHC of tumor biopsies revealed that the EMT phenotype was reversed. There was an inverse correlation between Snail inhibition and RKIP induction. The present findings demonstrate, for the first time, that NO donors (high concentrations) inhibit the EMT phenotype and suggest their potential therapeutic applications in vivo in patients. These studies also suggest that the NF-κB/Snail/RKIP circuitry is dysregulated in tumor cells and is responsible, in part, for the EMT phenotype. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1466.

Metastasis is the main cause of death in cancer patients. The underlying molecular mechanisms of the metastatic process and their regulation in different malignancies are unknown. Survival pathways, such as the constitutively activated NF-κB pathway, contribute to the initiation of metastasis, a process known as epithelial to mesenchymal transition (EMT). The EMT phenotype is characterized by several gene modifications such as inhibition of epithelial gene products (e.g. E-cadherin, cytokeratin 18) and upregulation of mesenchymal gene products (e.g. vimentin and fibronectin). In addition, the cells exhibit invasive properties. The metastasis-inducer transcription factor, Snail, is intimately involved in EMT and is transcriptionally regulated, in part, by NF-κB. A recent report demonstrated that Snail is transcriptionally regulated, in part, by the transcription factor Yin Yang 1 (YY1) and YY1 is downstream of NF-κB. We have reported that YY1 expression is a prognostic marker in prostate cancer and YY1 also regulates tumor cell resistance to apoptosis by cytotoxic therapeutics. Based on these findings, we hypothesized that the expression levels of YY1 in cancer may also regulate EMT directly and/or indirectly through its regulation of Snail transcription. This hypothesis was tested using the human metastatic prostate carcinoma cell lines, PC-3 and DU-145, that exhibit the EMT phenotype as models. The relationship between inhibition of YY1 and inhibition of EMT was established by several lines of evidence. Treatment of the tumor cells with the proteasome inhibitors, NPI-0052 or Bortezomib, inhibited NF-κB, Snail and YY1 activities and correlated with inhibition of EMT. The direct role of YY1 expression and activity in the regulation of EMT was shown by treating cells with YY1 siRNA and resulting in the inhibition of the EMT phenotype. In turn, inhibition of Snail resulted in upregulation of the expression of the metastasis-suppressor/immune surveillance cancer gene product, Raf-1 kinase inhibitor protein (RKIP). The upregulation of RKIP by YY1 siRNA was due to the inhibition of the RKIP transcription repressor Snail. These findings establish a novel role for YY1 in the regulation of EMT. The findings also establish the dysregulation of the circuitry NF-κB/Snail/YY1/RKIP in cancer and metastasis resulting in the initiation of EMT. Further, this dysregulation has also been recently reported by us to regulate tumor cell resistance to apoptotic-inducing stimuli. Therefore, this dysregulated circuitry and downstream regulated gene products may be considered as novel targets for the regulation of both metastasis and resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2290.

A subset of patients with B cell malignancies does not respond to conventional current cytotoxic therapies. Novel immunotherapeutic approaches have been considered such as TRAIL and agonist DR4/DR5 mAbs that are currently in phase I/II clinical trials in patients with a variety of tumor types. However, most tumors are not sensitive to TRAIL/agonist antibodies due to the development of a generalized resistance to apoptotic stimuli. Therefore, for these therapies to be effective it is a requisite that resistance is modified by the use of sensitizing agents that target cell survival anti-apoptotic pathways or, more selectively, specific gene products in the apoptotic pathways. Proteasome inhibitors such as bortezomib and the novel inhibitor NPI-0052 have shown both direct cytotoxicity and also sensitizing ability when used in combination with biologics and cytotoxic drugs. We have recently reported that treatment of TRAIL-resistance B-NHL cell lines with NPI-0052 (not toxic by itself) in combination with TRAIL resulted in significant potentiation of apoptosis and synergy was achieved. Sensitization was due, in part, to inhibition of NF-kB activity and induction of Raf-1 kinase inhibitor protein (RKIP) [Baritaki et al., J Immunol 80:6199, 2008]. The role of RKIP in TRAIL apoptosis by NPI-0052 in Ramos cells was examined as well as the mechanism of NPI-0052-induced expression of RKIP. Like NPI-0052, overexpression of RKIP in Ramos cells sensitized the cells to TRAIL apoptosis and, in contrast, treatment with siRNA RKIP reversed this sensitizing effect. A recent report by our collaborators [Beach et al., Oncogene 27:2243,2008] demonstrated that RKIP transcription is negatively regulated, in part, by the metastasis-inducer transcription factor Snail. Thus, we hypothesized that NPI-0052-induced upregulation of RKIP may be due to inhibition of Snail. Indeed, treatment of Ramos cells with NPI-0052 significantly downregulated Snail expression concomitantly with upregulation of RKIP. Treatment of Ramos cells with siRNA Snail resulted in upregulation of RKIP and sensitization to TRAIL apoptosis. In contrast, ectopic expression of Snail repressed RKIP expression. Further, ectopic expression of RKIP inhibited Snail expression while siRNA RKIP upregulated Snail. These findings revealed the existence of a regulatory RKIP-NF-kB-Snail-RKIP loop. NPI-0052-induced inhibition of Snail also resulted in inhibition of Snail-induced gene products involved in metastasis. These findings demonstrate that NPI-0052 regulates B-NHL sensitivity to TRAIL apoptosis via the inhibition of Snail and induction of RKIP. These findings support the potential therapeutic application of the combination of NPI-0052 and TRAIL in the treatment of patients with resistant B-NHL and other malignancies.

Objective and Rationale Raf-1 kinase inhibitor protein (RKIP) is a modulator of cell signaling and survival that functions as an endogenous inhibitor of multiple kinases, including kinases involved in the Raf-1/MEK/ERK and NF-kB pathways. RKIP has been identified as a metastasis suppressor gene and an immune surveillance cancer gene, since loss of RKIP protein expression has been associated with tumor progression, metastasis and escape from immune surveillance. Further, RKIP expression has been associated with prognostic significance in many cancers. Recently, we have demonstrated that induction of RKIP expression in tumors with low RKIP levels results in increased tumor cell sensitivity to immuno- or chemo-therapy via inhibition of the above pathways. However, multiple myeloma (MM) cells have been shown to express high RKIP levels compared to other tumors and still remain highly resistant to conventional cytotoxic therapies. These findings were unexpected and thus, it was plausible that the high level of RKIP expression was not functionally active. It has been reported that phosphorylation of RKIP at Ser-153 renders the cells inactive (Corbit et al., 2003, J Biol Chem 278:13061-8). Thus, we examined the expression and the phosphorylation status of the RKIP protein in several multiple myeloma cell lines and tissues and compared them with other cell lines with low RKIP expression. Hypothesis We hypothesized that MM tumor cells express high levels of the inactive phosphorylated RKIP protein which antagonizes the active non-phosphorylated RKIP form in the inhibition of the survival signaling pathways. Experimental Designs and Methods Multiple myeloma (IM-9, RPMI 8226, MM1S and U266 cell lines and fresh bone marry samples from MM patients), PC-3 prostatic carcinoma and Ramos B-NHL cell lines were examined for total and phosphorylated RKIP expression by IHC and Western Blot analyses. The total RKIP protein was significantly elevated in multiple myeloma cell lines compared to the prostate and B-NHL lines. The predominant RKIP form in patients’ multiple myeloma tumors was the phosphorylated RKIP protein with high nuclear localization, as assessed by IHC, while the phosphorylated RKIP levels in the non-myeloma tumors were relatively low. It has been reported that the phosphorylation of RKIP is mediated by protein kinase C (Corbit et al., 2003, J Biol Chem 278:13061–8). Additional studies in MM cell lines also revealed high expression of the zeta isoform of PKC (PKCζ), known to phosphorylate and inactivate RKIP. Conclusions and Implications The present findings demonstrate that the aberrant RKIP phosphorylation in MM tumors may result in the inhibition of the suppressive effect of RKIP on tumor survival signaling pathways. We postulate that the high expression of RKIP may be due to inhibition of proteasome degradation. The present findings also suggest that screening of RKIP levels and RKIP phosphorylation status in MM may be useful as prognostic factors of tumor cell response to anti-tumor therapies. (Baritaki and Huerta-Yepez contributed equally.)

Treatment of patients with B-NHL with a combination of rituximab and CHOP resulted in significant clinical response in greater than 90% of patients. The underlying mechanism of synergy achieved in-vivo is not clear; however, our recent studies with B-NHL cell lines revealed rituximab-induced inhibition of intracellular survival pathways that were responsible for reversal of resistance. The combination of rituximab and CHOP is associated with drug-induced toxicity, and thus, it is desirable to have a nontoxic agent that can replace CHOP with similar and improved clinical responses. The proteasome inhibitor, NPI-0052, has been shown to exert minimal toxicity, and induce cytotoxic activity against certain tumor cell lines and is currently in Phase I/II clinical trials as single agent and in combination with Zolinza against various cancers. We have reported that rituximab inhibits the NF-κB pathway concomitantly with the induction of Raf-1 kinase inhibitor protein (RKIP) and inhibition downstream of anti-apoptotic gene products (e.g. Bcl-2, Bc-lXL, Mcl-1, etc.). Likewise, NPI-0052 has also recently been shown to induce the expression of RKIP and inhibits downstream anti-apoptotic gene products. Based on the above findings, we hypothesized that treatment of resistant B-NHL cells with the combination of rituximab and NPI-0052 may result in the complementary induction of apoptosis through additive and/or synergistic effects as a result of inhibiting several survival and anti-apoptotic gene products regulated by NF-κB and induction of RKIP. This study was designed to test this hypothesis. Treatment of Ramos B-NHL cells with rituximab (20 μg/ml for 24 h) or NPI-0052 (20–40 nM) did not yield any significant apoptosis; however, the combination treatment resulted in significant potentiation of apoptosis and synergy was achieved. Treatment with rituximab or NPI-0052 alone resulted in inhibition of the NF-κB pathway, namely, IκBα and downstream BclXL and Mcl-1 and there was no activation of caspases. There was, however, significant induction of RKIP expression by each agent alone. The combination treatment resulted in additive effects with the activation of caspases 8, 9 and 3 and induction of apoptosis. The role of NF-κB inhibition by rituximab in synergy was corroborated with the use of the NF-κB inhibitor, DHMEQ, which sensitized the cells to apoptosis by NPI-0052. The role of RKIP induction in the regulation of apoptosis by NPI-0052 was demonstrated in cells over-expressing RKIP, which were sensitized to NPI-0052-induced apoptosis. In contrast, treatment with si-RNA RKIP reversed rituximab-induced sensitization to NPI-0052-induced apoptosis. Altogether, these findings reveal one mechanism by which rituximab sensitizes B-NHL cells to NPI-0052 apoptosis as the result of the concomitant induction of RKIP and inhibition of the NF-κB survival pathway. The findings also suggest the potential clinical application of rituximab and NPI-0052 in the treatment of patients with B-NHL with minimal toxicity. Furthermore, the findings suggest that agents that can induce RKIP may mimic rituximab in the sensitization to NPI-0052-induced apoptosis and their therapeutic application in patients who are not responsive to rituximab.

Patients with B-cell malignancies initially respond to conventional cytotoxic therapies. However, many patients experience relapses and recurrences. Novel therapeutics are being explored in the reversal of resistance such as TRAIL. TRAIL has been shown to be selectively cytotoxic to few tumors and both TRAIL and agonist TRAIL-R1 (DR4) and TRAIL-R2 (DR5) antibodies are currently being explored clinically in unresponsive cancer patients. We have recently found that treatment of TRAIL resistant B-NHL cell lines such as Ramos with the novel proteasome inhibitor NPI-0052 (Nereus Pharmaceuticals) sensitizes tumor cells to TRAIL-induced apoptosis. The concentration of NPI-0052 achieving optimal sensitization to TRAIL was in the range of 1–5 nM. In contrast 400 fold more VelcadeTM (bortezomib) was necessary to achieve a similar level of apoptosis. The mechanism of NPI-0052- mediated sensitization to TRAIL was examined. Treatment of Ramos with NPI-0052 inhibited significantly the constitutively activated NF-κB concomitantly with inhibition of the DR5 transcription repressor Yin Yang 1 (YY1) resulting in up-regulation of DR5 expression. The role of YY1 in NPI-0052-induced sensitization to TRAIL was corroborated by the use of YY1siRNA as treatment with the YY1siRNA mimicked NPI-0052 induced DR5 up-regulation and sensitization to TRAIL. Studies examining the inhibition of the NF-κB pathway by NPI-0052 revealed a novel finding demonstrating the induced expression of the metastatic suppressor gene, Raf-1 kinase inhibitory protein (RKIP). RKIP has been reported to inhibit NF-κB activity and, thus, its induction by NPI-0052 contributed to the inhibition of NF-κB activity. The role of RKIP in NPI-0052 induced sensitization to TRAIL was corroborated by the use of RKIPsiRNA and transfectants mimicked NPI-0052 mediated inhibition of YY1, DR5 upregulation and sensitization to TRAIL mediated apoptosis. The above findings suggest that NPI-0052-induced inhibition of NF-κB results at least from two distinct mechanisms, namely, inhibition of p-IκBα degradation by the proteasome and by the induction of RKIP expression. The apoptosis induced by combination of NPI-0052 and TRAIL was the result of the activation of the type II mitochondrial pathway. It has been reported that the RKIP levels are reduced in many tumors (e.g. breast, prostate, ovarian) and the expression levels are of prognostic significance. Hence, the present findings demonstrating NPI-0052 induced RKIP expression may be therapeutically relevant in the prevention of metastasis. In addition, NPI-0052 should facilitate the reversal of tumor resistance when used in combination with subtoxic levels of cytotoxics.